Confocal imaging methods and apparatus

• US 20070114362A1
• Filed: 11/23/2005
• Published: 05/24/2007
• Est. Priority Date: 11/23/2005
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Triener, Alexander, Wang, Mark, Che, Diping, Feng, Wenyi, Kain, Robert, Kotseroglou, Theofilos
• Granted Patent: US 7,329,860 B2
• Time in Patent Office: Days
• US Class Current: 250/208.100
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/0032 Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06
• CPC Class Codes: G02B 21/004 fixed arrays, e.g. switchable aperture arrays

Abstract

The invention provides imaging apparatus and methods useful for obtaining a high resolution image of a sample at rapid scan rates. A rectangular detector array having a horizontal dimension that is longer than the vertical dimension can be used along with imaging optics positioned to direct a rectangular image of a portion of a sample to the rectangular detector array. A scanning device can be configured to scan the sample in a scan-axis dimension, wherein the vertical dimension for the rectangular detector array and the shorter of the two rectangular dimensions for the image are in the scan-axis dimension, and wherein the vertical dimension for the rectangular detector array is short enough to achieve confocality in a single axis.

Claims

1. An imaging apparatus comprising:

(a) a radiation source positioned to send excitation radiation to at least a portion of a sample region;

(b) a rectangular detector array;

(c) imaging optics positioned to direct a rectangular image of said portion to said rectangular detector array; and

(d) a scanning device configured to scan said sample region in a scan-axis dimension, whereby the portion of said sample region that forms a rectangular image at said rectangular detector array is changed,

wherein the shorter of the two rectangular dimensions for said rectangular detector array and the shorter of the two rectangular dimensions for said image are in said scan-axis dimension, and

wherein said shorter of the two rectangular dimensions for said rectangular detector array is short enough to achieve confocality in a single axis of said rectangular detector array, wherein said single axis is said shorter of the two rectangular dimensions for said rectangular detector array.

2. The apparatus of claim 1, wherein the ratio of said shorter of the two rectangular dimensions for said rectangular detector array and the product of the Rayleigh resolution of the imaging optics multiplied by the magnification of the imaging optics is in the range of 0.1 to 10.

3. The apparatus of claim 1, further comprising a line generator positioned to receive excitation radiation from said radiation source and to send a radiation line to said sample region.

4. The apparatus of claim 3, further comprising an objective positioned to receive said radiation line therethrough to illuminate said sample region.

5. The apparatus of claim 4, wherein said imaging optics comprise said objective, wherein said objective is further positioned to collect radiation emanating from said sample region, wherein said radiation emanating from said sample region forms said rectangular image that is directed to said rectangular detector array.

6. The apparatus of claim 5, further comprising a beam splitter positioned to separate said radiation line from said radiation emanating from said sample region and to direct said radiation emanating from said sample region to the rectangular detector array.

7. The apparatus of claim 4, wherein said objective has an external pupil positioned to receive said radiation line therethrough to illuminate said sample region.

8. The apparatus of claim 4, further comprising a first expander positioned to receive excitation radiation from said radiation source and to send an expanded beam of said radiation to said line generator.

9. The apparatus of claim 8, further comprising a second expander positioned to receive said excitation radiation from said line generator and send an expanded beam of said radiation to said objective, wherein said second expander is further configured to decrease the field angle of said radiation line.

10. The apparatus of claim 4, wherein said objective has a property selected from the group consisting of color correction, high numerical aperture, telecentricity, and afocality at the backplane.

11. The apparatus of claim 3, wherein said line generator has a full fan angle of six degrees and is configured to receive an input beam having a diameter of at most 4 mm.

12. The apparatus of claim 3, wherein said line generator further comprises a cylindrical micro-lens array, one-dimensional diffuser having an angular uniformity, aspheric refractive lens, diffractive element or Powell lens.

13. The apparatus of claim 3, wherein said line generator further comprises a diffractive element to generate a diffraction-limited line with uniform intensity distribution.

14. The apparatus of claim 3, wherein the shorter of two rectangular dimensions for said radiation line is short enough to achieve confocality in a single axis of said rectangular detector array, wherein said single axis is said shorter of the two rectangular dimensions for said rectangular detector array.

15. The apparatus of claim 3, wherein the ratio of the shorter of two rectangular dimensions for said radiation line to the quotient of said shorter of the two rectangular dimensions divided by the magnification of the imaging optics is in the range of 0.1 to 10.

16. The apparatus of claim 3, wherein the ratio of the shorter of two rectangular dimensions for said radiation line to the Rayleigh resolution of the imaging optics is in the range of 0.1 to 10.

17. The apparatus of claims 3, wherein the ratio of the 1/e{grave over ( )}2 width of the shorter of two rectangular dimensions for said radiation line to the quotient of said shorter of the two rectangular dimensions for said rectangular detector array divided by the magnification of the imaging optics is in the range of 0.5 to 2.

18. The apparatus of claim 1, further comprising a projection lens positioned to collect radiation emanating from said sample region, wherein said radiation emanating from said sample region forms said rectangular image that is directed to said rectangular detector array.

19. The apparatus of claim 1, further comprising a band pass filter positioned to collect radiation emanating from said sample region, wherein said radiation emanating from said sample region forms said rectangular image that is directed to said rectangular detector array.

20. The apparatus of claim 1, further comprising an emission filter positioned to collect radiation emanating from said sample region, wherein said radiation emanating from said sample region forms said rectangular image that is directed to said rectangular detector array.

21. The apparatus of claim 1, further comprising a translation stage positioned to provide a sample to said sample region.

22. The apparatus of claim 21, wherein said translation stage is configured to move said sample in said scan-axis dimension.

23. The apparatus of claim 21, further comprising a microarray supported by said translation stage, whereby said array is provided to said sample region.

24. The apparatus of claim 1, wherein said rectangular detector array is configured for TDI (Time Delay Integration) operation.

25. The apparatus of claim 1, wherein said rectangular detector array comprises a line scan CCD camera, CMOS detector array, avalanche photodiode (APD) array, or Geiger-mode photon counter array.

26. The apparatus of claim 1, wherein the aspect ratio of said rectangular detector is greater than 20.

27. The apparatus of claim 1, wherein said radiation source comprises at least one laser.

28. The apparatus of claim 1, comprising multiple collection arms positioned to collect radiation emanating from said sample region, wherein said radiation emanating from said sample region forms multiple rectangular images that are directed to multiple rectangular detector arrays.

29. The apparatus of claim 1, wherein said apparatus is configured to obtain an image of said sample comprising a Rayleigh resolution between 0.2 and 10 micrometers.

30. A method of obtaining an image of a sample, comprising

(a) contacting at least a first portion of a sample with excitation radiation under conditions wherein radiation is emanated from said first portion;

(b) directing said radiation emanated from said first portion to form a rectangular image of said first portion at a rectangular detector array; and

(c) scanning said sample region in a scan-axis dimension, thereby repeating steps (a) and (b) to form a rectangular image of a second portion of said sample at said rectangular detector array,

wherein the shorter of the two rectangular dimensions for said rectangular detector array and the shorter of the two rectangular dimensions for said images are in said scan-axis dimension, and

wherein said shorter of the two rectangular dimensions for said rectangular detector array is short enough to achieve confocality in a single axis of said rectangular detector array, wherein said single axis is said shorter of the two rectangular dimensions for said rectangular detector array.

31. The method of claim 30, wherein the ratio of said shorter of the two rectangular dimensions for said rectangular detector array and the product of the Rayleigh resolution of said rectangular image multiplied by the magnification of the rectangular image is in the range of 0.1 to 10.

32. The method of claim 30, wherein said excitation radiation that contacts at least a portion of said sample comprises a radiation line.

33. The method of claim 32, wherein the shorter of two rectangular dimensions for said radiation line is short enough to achieve confocality in a single axis of said rectangular detector array, wherein said single axis is said shorter of the two rectangular dimensions for said rectangular detector array.

34. The method of claim 32, wherein the ratio of the shorter of two rectangular dimensions for said radiation line to the quotient of said shorter of the two rectangular dimensions for said rectangular detector array divided by the magnification is in the range of 0.1 to 10.

35. The method of claim 30, wherein the ratio of the 1/e{grave over ( )}2 width the shorter of two rectangular dimensions for said radiation line to the quotient of said shorter of the two rectangular dimensions for said rectangular detector array divided by the magnification is in the range of 0.5 to 2.

36. The method of claim 30, wherein said scanning said sample comprises moving said sample, thereby changing the relative positions of said rectangular image and said rectangular detector array in said scan-axis dimension.

37. The method of claim 30, wherein said scanning comprises TDI (Time Delay Integration).

38. The method of claim 30, wherein all pixel elements in the shorter of the two rectangular dimensions for said rectangular detector array are collected in a common bin and read out as a single value.

39. The method of claim 30, wherein said excitation radiation comprises radiation in a range selected from the group consisting of UV radiation, VIS radiation and IR radiation.

40. The method of claim 30, further comprising storing a data representation of said image of said sample in a computer readable memory.

41. The method of claim 40, further comprising displaying a graphical representation of said image of said sample on a monitor operably connected to said computer readable memory.

42. The method of claim 30, wherein said sample comprises a microarray having a plurality of individual sites.

43. The method of claim 42, wherein said individual sites are separated by a distance in the range of 0.1 to 50 micrometers.

44. The method of claim 43, further comprising distinguishing said individual sites.

45. The method of claim 30, wherein said image of said sample comprises a Rayleigh resolution between 0.2 and 10 micrometers.

46. A method of configuring a scanner to achieve confocality in a single axis, comprising,

(a) providing an apparatus comprising

(i) a radiation source positioned to send excitation radiation to at least a portion of a sample region;

(ii) a rectangular detector array;

(iii) imaging optics positioned to direct a rectangular image of said portion to said rectangular detector array; and

(iv) a scanning device configured to scan said sample region in a scan-axis dimension, whereby the portion of said sample region that forms a rectangular image at said rectangular detector array is changed,

wherein the shorter of the two rectangular dimensions for said rectangular detector array and the shorter of the two rectangular dimensions for said image are in said scan-axis dimension; and

(b) positioning said rectangular detector array or said imaging optics to restrict said shorter of the two rectangular dimensions for said rectangular detector array to be short enough to achieve confocality in a single axis of said rectangular detector array, wherein said single axis is said shorter of the two rectangular dimensions for said rectangular detector array.